This work was supported by R01 NS105807/NS/NINDS NIH HHS/United States and CURE based on a grant CURE received from the United States Army Medical Research and Materiel Command, Department of Defense (DoD), through the Psychological Health and Traumatic Brain Injury Research Program under Award No. W81XWH-15-2-0069. Ivan Zuidhoek is greatly appreciated for proofreading the manuscript.

All animal procedures described in this protocol were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of Virginia Tech and in compliance with the National Institutes of Health&#39;s &#39;Guide for the Care and Use of Laboratory Animals&#39;.
1. Animal handling protocol
NOTE: This protocol is intended to habituate animals ordered from a vendor to the facility after arrival and to condition them to being handled by the experimenter. ...

<ol>
	<li>Christensen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+risk+of+epilepsy+after+traumatic+brain+injury+in+children+and+young+adults:+a+population-based+cohort+study.">Long-term risk of epilepsy after traumatic brain injury in children and young adults: a population-based cohort study.</a> Lancet. 373 (9669), 1105-1110 (2009).</li><li>Lowenstein, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epilepsy+after+head+injury:+an+overview.">Epilepsy after head injury: an overview.</a> Epilepsia. 50, 4-9 (2009).</li><li>Ferguson, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population-based+study+of+risk+of+epilepsy+after+hospitalization+for+traumatic+brain+injury.">A population-based study of risk of epilepsy after hospitalization for traumatic brain injury.</a> Epilepsia. 51 (5), 891-898 (2010).</li><li>Abou-Abbass, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+clinical+characteristics+of+traumatic+brain+injury+in+Lebanon:+A+systematic+review.">Epidemiology and clinical characteristics of traumatic brain injury in Lebanon: A systematic review.</a> Medicine (Baltimore). 95 (47), 5342 (2016).</li><li>Management of Concussion/mTBI Working Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VA/DoD+Clinical+Practice+Guideline+for+Management+of+Concussion/Mild+Traumatic+Brain+Injury.">VA/DoD Clinical Practice Guideline for Management of Concussion/Mild Traumatic Brain Injury.</a> The Journal of Rehabilitation Research and Development. 46 (6), 1-68 (2009).</li><li>Piccenna, L., Shears, G., O'Brien, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+post-traumatic+epilepsy:+An+evidence+review+over+the+last+5+years+and+future+directions.">Management of post-traumatic epilepsy: An evidence review over the last 5 years and future directions.</a> Epilepsia Open. 2 (2), 123-144 (2017).</li><li>Loscher, W., Brandt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+or+modification+of+epileptogenesis+after+brain+insults:+experimental+approaches+and+translational+research.">Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research.</a> Pharmacological Reviews. 62 (4), 668-700 (2010).</li><li>Ostergard, T., Sweet, J., Kusyk, D., Herring, E., Miller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+post-traumatic+epilepsy.">Animal models of post-traumatic epilepsy.</a> Journal of Neuroscience Methods. 272, 50-55 (2016).</li><li>Shandra, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repetitive+Diffuse+Mild+Traumatic+Brain+Injury+Causes+an+Atypical+Astrocyte+Response+and+Spontaneous+Recurrent+Seizures.">Repetitive Diffuse Mild Traumatic Brain Injury Causes an Atypical Astrocyte Response and Spontaneous Recurrent Seizures.</a> Journal of Neuroscience. 39 (10), 1944-1963 (2019).</li><li>Foda, M. A., Marmarou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+diffuse+brain+injury+in+rats.+Part+II:+Morphological+characterization.">A new model of diffuse brain injury in rats. Part II: Morphological characterization.</a> Journal of Neurosurgery. 80 (2), 301-313 (1994).</li><li>Marmarou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+diffuse+brain+injury+in+rats.+Part+I:+Pathophysiology+and+biomechanics.">A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics.</a> Journal of Neurosurgery. 80 (2), 291-300 (1994).</li><li>Paxinos, G., Keith, B. J., Franklin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2007).</li><li>Shandra, O., Robel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+Manipulating+Astrocyte+Function+In+Vivo+in+the+Context+of+CNS+Injury.">Imaging and Manipulating Astrocyte Function In Vivo in the Context of CNS Injury.</a> Methods in Molecular Biology. 1938, 233-246 (2019).</li><li>Pitkanen, A., Immonen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epilepsy+related+to+traumatic+brain+injury.">Epilepsy related to traumatic brain injury.</a> Neurotherapeutics. 11 (2), 286-296 (2014).</li><li>Kharatishvili, I., Nissinen, J. P., McIntosh, T. K., Pitkanen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+posttraumatic+epilepsy+induced+by+lateral+fluid-percussion+brain+injury+in+rats.">A model of posttraumatic epilepsy induced by lateral fluid-percussion brain injury in rats.</a> Neuroscience. 140 (2), 685-697 (2006).</li><li>Pitkanen, A., Bolkvadze, T., Immonen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-epileptogenesis+in+rodent+post-traumatic+epilepsy+models.">Anti-epileptogenesis in rodent post-traumatic epilepsy models.</a> Neuroscience Letters. 497 (3), 163-171 (2011).</li><li>Gades, N. M., Danneman, P. J., Wixson, S. K., Tolley, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+magnitude+and+duration+of+the+analgesic+effect+of+morphine,+butorphanol,+and+buprenorphine+in+rats+and+mice.">The magnitude and duration of the analgesic effect of morphine, butorphanol, and buprenorphine in rats and mice.</a> Journal of the American Association for Laboratory Animal Science. 39 (2), 8-13 (2000).</li></ol>

In contrast to CCI and FPI models inducing either focal or combination of focal and diffuse injury, the model of repetitive diffuse TBI described in this protocol allows for the induction of diffuse injury in the absence of focal brain injury and does not require scalp or cranial openings and the associated inflammation. An added benefit of the absence of craniectomy in this model is that it allows to not only implant the electrodes for chronic continuous EEG recording, but also the creation of a thinned-skull cranial wi...

Every year TBI affects an estimated 60 million people worldwide. Impacted individuals are at higher risk of developing epilepsy, which can manifest years after the initial injury. Though severe TBIs are associated with a higher risk of epilepsy, even mild TBI increases an individual&rsquo;s chance of developing epilepsy1,2,3,4. All TBIs can be classified as focal, diffuse, or a combination of both. Diffuse brain injury, present in many if not all TBIs, is a result of brain tissues of different densities shearing against each other due to acceleration-deceleration and rotational forces. By definition, diffuse injury only occurs in isolation in mild/concussive non-penetrating brain injury, in which no brain lesions are visible on computed tomography scans5.
There are currently two critical problems in the management of patients who have, or are at risk of, developing post-traumatic epilepsy (PTE). The first is that once PTE has manifested, seizures are resistant to available anti-epileptic drugs (AEDs)6. Secondly, AEDs are equally ineffective at preventing epileptogenesis, and there are no effective alternative therapeutic approaches. In order to address this deficit and find better therapeutic targets and candidates for treatment, it will be necessary to explore new cellular and molecular mechanisms at the root of PTE6.
One of the prominent features of post-traumatic epilepsy is the latent period between the initial traumatic event and the onset of spontaneous, unprovoked, recurrent seizures. The events that occur within this temporal window are a natural focus for researchers, because this time window might allow treatment and prevention of PTE altogether. Animal models are most commonly used for this research because they offer several distinct benefits, not the least of which is that continuous monitoring of human patients would be both impractical and costly over such potentially long spans of time. Additionally, cellular and molecular mechanisms at the root of epileptogenesis can only be explored in animal models.
Animal models with spontaneous post-traumatic seizures and epilepsy are preferred over models where seizures are induced after TBI by less physiologically relevant means, such as by chemoconvulsants or electric stimulation acutely, chronically, or by kindling. Spontaneous post-traumatic seizure models test how TBI modifies the healthy brain network leading to epileptogenesis. Studies using additional stimulation after TBI assess how exposure to TBI reduces seizure threshold and affects susceptibility to seizures. The advantages of animal models with seizures induced chemically or with electric stimulation are in testing the specific mechanisms of refractoriness to AEDs and the efficacy of existing and novel AEDs. Yet, the degree of relevance and translation of these data to humans may be ambiguous7 due to the following: 1) seizure mechanisms may be different from those induced by TBI alone; 2) not all of these models lead to spontaneous seizures7; 3) lesions created by the convulsant agent itself, with the cannula required for its delivery, or by stimulating electrode placement in depth structures (e.g., the hippocampus or amygdala) can already cause increased seizure susceptibility and even hippocampal epileptiform field potentials7. Furthermore, some convulsant agents (i.e., kainic acid) produce direct hippocampal lesions and sclerosis, which is not typical after diffuse TBI.
Until recently, only two animal models of post-traumatic epilepsy existed: controlled cortical impact (CCI, focal) or fluid percussion injury (FPI, focal and diffuse)8. Both models result in large focal lesions alongside tissue loss, hemorrhage, and gliosis in rodents8. These models mimic post-traumatic epilepsy induced by large focal lesions. A recent study demonstrated that repeated (3x) diffuse TBI is sufficient for the development of spontaneous seizures and epilepsy in mice even in the absence of focal lesions9, adding a third rodent PTE model with confirmed spontaneous recurrent seizures. This new model mimics cellular and molecular changes induced by diffuse TBI, better representing the human population with mild, concussive TBIs. In this model, the latent period of three weeks or more before seizure onset and the emergence of late, spontaneous, recurrent seizures allows for investigating the root causes of post-traumatic epileptogenesis, testing the efficacy of preventive approaches and new therapeutic candidates after seizure onset, and has potential for the development of biomarkers of post-traumatic epileptogenesis because approximately half of the animals develop post-traumatic epilepsy.
The choice of animal model for the study of post-traumatic epilepsy depends on the scientific question, the type of brain injury investigated, and what tools will be used to determine the underlying cellular and molecular mechanisms. Ultimately, any model of post-traumatic epilepsy must demonstrate both the emergence of spontaneous seizures after TBI and an initial latency period in a subset of TBI animals, because not all patients who incur a TBI go on to develop epilepsy. To do this, electroencephalography (EEG) with simultaneous video acquisition is used in this protocol. Understanding the technical aspects behind data acquisition hardware and approaches is critical for accurate data interpretation. The critical hardware aspects include the type of recording system, type of electrodes (screw or wire lead) and material they are made of, synchronized video acquisition (as part of the EEG system or third party), and properties of the computer system. It is imperative to set the appropriate acquisition parameters in any type of system depending on study goal, EEG events of interest, further analysis method, and sustainability of data storage. Lastly, the method of electrode configuration (montage) must be considered, as each has advantages and disadvantages and will affect the data interpretation.
This protocol details how to use the modified Marmarou weight drop model10,11 to induce diffuse injury resulting in spontaneous, unprovoked, recurrent seizures in mice, describes surgical approaches to acquire a single- and multi-channel continuous, and synchronized video EEG using monopolar, bipolar, or mixed montage.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.10&#34; screw</td>
			<td>Pinnacle Technology Inc., KS, USA</td>
			<td>8209</td>
			<td>0.10 inch long stainless steel</td>
		</tr>
		<tr>
			<td>0.10&#34; screw</td>
			<td>Pinnacle Technology Inc., KS, USA</td>
			<td>8403</td>
			<td>0.10 inch long with pre-soldered wire lead</td>
		</tr>
		<tr>
			<td>0.12&#34; screw</td>
			<td>Pinnacle Technology Inc., KS, USA</td>
			<td>8212</td>
			<td>0.12 inch long stainless steel</td>
		</tr>
		<tr>
			<td>1EEG headmount</td>
			<td>Invitro1 (subsidiary of Plastics One), VA, USA</td>
			<td>MS333/8-A/SPC</td>
			<td>3 individually Teflon-insulated platinum iridium wire electrodes (twisted or untwisted, 0.005 inch diameter) extending below threaded plastic pedestal</td>
		</tr>
		<tr>
			<td>2EEG/1EMG headmount</td>
			<td>Pinnacle Technology Inc., KS, USA</td>
			<td>8201</td>
			<td>2EEG/1EMG channels</td>
		</tr>
		<tr>
			<td>3% hydrogen peroxide</td>
			<td></td>
			<td></td>
			<td>Pharmacy</td>
		</tr>
		<tr>
			<td>3EEG headmount</td>
			<td>Pinnacle Technology Inc., KS, USA</td>
			<td>8235-SM-C</td>
			<td>custom 6-Pin Connector for 3EEG channels</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Par Pharmaceuticals, Cos. Inc., Spring Valley, NY, USA</td>
			<td>060969</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Par Pharmaceuticals, Cos. Inc., Spring Valley, NY, USA</td>
			<td>060969</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Harlan/Envigo Laboratories Inc</td>
			<td></td>
			<td>male, 12-16 weeks old</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td>male, 12-16 weeks old</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Zoetis Services LLC, Parsippany, NJ, USA</td>
			<td>026357</td>
			<td>NOTE: this drug is added during weight drop only if stereotactic electrode implantation will be performed on the same day</td>
		</tr>
		<tr>
			<td>Chlorhexidine antiseptic</td>
			<td></td>
			<td></td>
			<td>Pharmacy</td>
		</tr>
		<tr>
			<td>Dental cement and solvent kit</td>
			<td>Stoelting Co., USA</td>
			<td>51459</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Foredom</td>
			<td>HP4-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill bit</td>
			<td>Meisinger USA, LLC, USA</td>
			<td>HM1-005-HP</td>
			<td>0.5 mm, Round, 1/4, Steel</td>
		</tr>
		<tr>
			<td>Dry sterilizer</td>
			<td>Cellpoint Scientific, USA</td>
			<td></td>
			<td>Germinator 500</td>
		</tr>
		<tr>
			<td>EEG System 1</td>
			<td>Biopac Systems, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EEG System 2</td>
			<td>Pinnacle Technology Inc., KS, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol &#8805;70%</td>
			<td>VWR, USA</td>
			<td>71001-652</td>
			<td>KOPTEC USP, Biotechnology Grade (140 Proof)</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Pro Labs Ltd, USA</td>
			<td></td>
			<td>Puralube Vet Ointment Sterile Ocular Lubricant available in general online stores and pharmacies</td>
		</tr>
		<tr>
			<td>Fluriso liquid for inhalation anesthesia</td>
			<td>MWI Veterinary Supply Co., USA</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal product</td>
			<td>Church &#38; Dwight Co., Inc., USA</td>
			<td></td>
			<td>Nair cream</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>MWI Veterinary Supply Co., USA</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine surgical solution</td>
			<td>Purdue Products, USA</td>
			<td>004677</td>
			<td>Betadine</td>
		</tr>
		<tr>
			<td>Rimadyl/Carprofen</td>
			<td>Zoetis Services LLC, Parsippany, NJ, USA</td>
			<td>026357</td>
			<td></td>
		</tr>
		<tr>
			<td>Solder</td>
			<td></td>
			<td></td>
			<td>Harware store</td>
		</tr>
		<tr>
			<td>Soldering iron</td>
			<td>Weller, USA</td>
			<td>WP35</td>
			<td>ST7 tip, 0.8mm</td>
		</tr>
		<tr>
			<td>Stainless steel disc</td>
			<td></td>
			<td></td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Sterile cotton swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze pads</td>
			<td>Fisher Scientific, USA</td>
			<td>22362178</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile poly-lined absorbent towels pads</td>
			<td>Cardinal Health, USA</td>
			<td>3520</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive</td>
			<td>3M Animal Care Products, USA</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

inducing post-traumatic epilepsy in a mouse model of repetitive diffuse traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of direct mechanical forces, sometimes penetrating through the cranium, creating a direct lesion in the brain tissue. These are visible during brain imaging as areas with contusion, laceration, and hemorrhage. Focal lesions induce neuronal death and glial scar formation and are present in 20%&minus;25% of all people who incur a TBI. However, in the majority of TBI cases, injury is caused by acceleration-deceleration forces and subsequent tissue shearing, resulting in nonfocal, diffuse damage. A subpopulation of TBI patients continues to develop post-traumatic epilepsy (PTE) after a latency period of months or years. Currently, it is impossible to predict which patients will develop PTE, and seizures in PTE patients are challenging to control, necessitating further research. Until recently, the field was limited to only two animal/rodent models with validated spontaneous post-traumatic seizures, both presenting with large focal lesions with massive tissue loss in the cortex and sometimes subcortical structures. In contrast to these approaches, it was determined that diffuse TBI induced using a modified weight drop model is sufficient to initiate development of spontaneous convulsive and non-convulsive seizures, even in the absence of focal lesions or tissue loss. Similar to human patients with acquired post-traumatic epilepsy, this model presents with a latency period after injury before seizure onset. In this protocol, the community will be provided with a new model of post-traumatic epilepsy, detailing how to induce diffuse non-lesional TBI followed by continuous long-term video-electroencephalographic animal monitoring over the course of several months. This protocol will detail animal handling, the weight drop procedure, the electrode placement for two acquisition systems, and the frequent challenges encountered during each of the steps of surgery, postoperative monitoring, and data acquisition.

The protocol outlined here describes the method for induction of a diffuse injury in isolation (e.g., in the absence a focal lesion) using a mouse model of repetitive diffuse TBI (Figure 1). Figure 1A depicts the weight drop device and its components (Figure 1A, a1&#8722;a5) used for induction of TBI in this model and crucial steps during the procedure (Figure 1...

Watch this Scientific Journal Video about Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury at JoVE.com

Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury

This systematic protocol describes a new animal model of post-traumatic epilepsy after repetitive mild traumatic brain injury. The first part details steps for traumatic brain injury induction using a modified weight drop model. The second part provides instructions on the surgical approach for single- and multi-channel electroencephalographic data acquisition systems.

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of ...

Our protocol is the first to detail a mouse model of post-traumatic epilepsy induced by mild traumatic brain injury. The use of a mild diffused traumatic brain injury model mimics an important aspect of the pathology of human traumatic brain injury which almost always has a diffused component. In order to develop biomarkers and treatments, research using models that appropriately reproduce aspects of human pathology is needed.Our traumatic brain injury model also has the potential to provide insight into the mechanisms that cause impairment of brain function after mild traumatic brain injury or concussion. After confirming a lack of response to pain reflex, place the mouse onto a foam pad and position the head of the mouse under the weight drop tube. Place a flat 1.3 centimeter diameter one millimeter thick 880 milligram weight stainless steel disc in the center of the animal's head between the eyes and the ears.Remove the pin in the weight drop tube to release the 100 gram weight rod from a height of 50 centimeters. To induce a sham injury in the control mice, remove the weight rod from the tube to prevent accidental release of the pin during weight drop. After weight drop induction, place the animal on its back on a heating pad covered with a sterile poly lined absorbent towel with monitoring until full recumbency.Then place the mouse in a clean cage that has been warmed on a heating pad with recovery gel and a few moistened chow pieces for 45 minutes. To prepare the surgical field for EEG electrode implantation, place a temperature sensor onto the pad and place the re-anesthetized brain injured mouse onto a sterile drape on top of the heating pad on the stereotactic apparatus. After fixing the head in place with ear bars, apply lubricating ointment to the animal's eyes and use depilatory cream to remove the hair from the scalp.Disinfect the exposed skin with three 20-second alternating swabs of povidone iodine surgical antiseptic solution and 70%ethanol in a circular motion. After removing the scalp, apply small hemostats on the opened skin borders to expand the exposed area. Next, use the scalpel to gently remove the periosteum and swab the cranium with hydrogen peroxide.When the cranium is cleared of tissue and exhibits a whitish appearance, dry the bone with a sterile gauze or swab. For electrode implantation, use a high-speed drill with a round 0.5 millimeter steel bit at 5, 000 to 6, 000 rotations per minute to create six burr holes for the screw electrodes. When all of the holes have been drilled, place the six screw electrodes into the burr holes and mix a half scoop of dental cement powder with several drops of solvent.Use a spatula to stir until the dental cement mixture is tacky but malleable and stiff enough to be properly condensed when placed on the animal's cranium. Apply the cement over the entire exposed surface of the cranium and each screw electrode. After one to two minutes, turn on the soldering iron and place the three EEG head mount into a stereotactic holder arm.Position the head mount over the skull so that the six wire lead positions match the position of the wire lead of each screw electrode. Lower the device so that its ventral part rests on top of the dental cement. Twist the wire of each lead from each of the screw electrodes with the corresponding wire lead of the head mount, solder each twisted pair of wire for proper signal conduction and use scissors to trim any excess wire.Bend each soldered pair of wire leads around the head mount. When all of the pairs have been bent, cover all of the wire with fresh dental cement leaving only the black portion of the head mount exposed. Release the hemostats holding the skin flaps and apply chlorhexidine antiseptic to the area around the implant to avoid infection.Then transfer the animal from the apparatus to a balance to measure the animal's weight as a reference for future monitoring before placing the animal in a clean cage on a warm heating pad with monitoring, recovery gel, and a few moistened chow pieces. In this figure, a spontaneous seizure in a mouse 97 days after repeated weight drop traumatic brain injury recorded using a three EEG head mount configured as demonstrated in this video protocol is shown. Here, a spontaneous non-convulsive electrographic seizure recorded in a mouse 65 days after repeated weight drop traumatic brain injury is shown.In this figure, a spontaneous seizure in a mouse 23 days after repeated weight drop traumatic brain injury was recorded using a one EEG head mount. The spectral power of a typical seizure indicates the highest density to be in the range of 10 to 40 hertz with a peak at 15 hertz. This protocol demonstrates the timelines for seizure onset in animals after repetitive weight drop traumatic brain injury demonstrating the incidence of seizure clustering in some animals and emphasizing the importance of acquiring continuous rather than intermittent recordings.Proper placement of the steel disc, careful implantation of the screw electrodes and careful soldering of the wire leads are all crucial for a successful long-term experiment. After EEG monitoring, animals with brain injury that develop seizures can be stratified from those that do not for subsequent analysis of their cellular, molecular, and physiological differences. To stratify animals with post-traumatic epilepsy from those that incur traumatic brain injury but do not develop seizures is critical for the development of interventions and biomarkers.

inducing-post-traumatic-epilepsy-mouse-model-repetitive-diffuse

Research

JoVE Journal

Behavior

10.2K visualizações.  Fralin Biomedical Research Institute at Virginia Tech Carilion. Nosso protocolo é o primeiro a detalhar um modelo de camundongo de epilepsia pós-traumática induzida por lesão cerebral traumática leve. O uso de um modelo de lesão cerebral traumática leve e difusa imita um aspecto importante da patologia da lesão cerebral traumática humana que quase sempre tem um componente difundido. Para desenvolver biomarcadores e tratamentos, são necessárias pesquisas utilizando modelos que reproduzem adequadamente aspectos da patologia humana.Nosso mo...